Hierarchical detection of diverse Clade II (atypical) nosZ genes using new primer sets for classical- and multiplex PCR array applications

Authors: Chee Sanford, Joanne; Connor, Lynn; KRICHELS, ALEXANDER - University Of Illinois; YANG, WENDY - University Of Illinois; SANFORD, ROBERT - University Of Illinois

Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/25/2020
Publication Date: 3/29/2020
Citation: Chee-Sanford, J.C., Connor, L.M., Krichels, A., Yang, W.H., Sanford, R.A. 2020. Hierarchical detection of diverse Clade II (atypical) nosZ genes using new primer sets for classical- and multiplex PCR array applications. Journal of Microbiological Methods. 172:105908. https://doi.org/10.1016/j.mimet.2020.105908.
DOI: https://doi.org/10.1016/j.mimet.2020.105908

Interpretive Summary: Nitrous oxide (N2O) is reduced biologically in a reaction catalyzed by the enzyme nitrous oxide reductase (NosZ). This activity was traditionally thought to be associated only with denitrifying microorganisms as the last sequential step in the reduction of nitrate (NO3-) to dinitrogen (N2), whereby mitigating N2O release to the atmosphere but responsible for major losses of N from agricultural soils. The gene responsible is nosZ, now discovered to comprise two main groups, Clade I and Clade II; the latter found in highly diverse bacteria that include both non-denitrifiers and denitrifiers and dominate in many soil environments. Consequently, the biological N2O sink in soils have largely been underestimated, with studies to date hampered by the lack of available molecular tools to aid detection of the wide diversity of bacteria with Clade II nosZ. The novel approach of this research was to conduct a comprehensive analysis of nosZ genes from the genomes of known bacteria and actual sequences retrieved from soil environments, then design and validate new PCR primers that target specific subgroups of Clade II nosZ. Seven of ten subgroups were effectively targeted, demonstrating applicability to current gene sequencing technology and quantitative gene analyses. The results of this study provide better research tools to fully characterize the microbial community of N2O reducers in natural systems, and further, allow transcriptomic studies to identify the active populations responsible for N2O reduction. The impact from such studies will fill major gaps in microbial N-cycling and advance our ability to better predict the fate of N2O in soils.

Technical Abstract: The reduction of nitrous oxide (N2O) to N2 represents the key terminal step in canonical denitrification. Nitrous oxide reductase (NosZ), the enzyme associated with this biological step, however, is not always affiliated with denitrifying microorganisms. Such organisms were shown recently to possess an atypical (Clade II) nosZ gene, in contrast to typical (Clade I) nosZ harbored in more commonly studied denitrifiers. Subsequent phylogenetic analyses have shown that Clade II NosZ are affiliated with a much broader diversity of microorganisms than those with Clade I NosZ, the former including both non-denitrifiers and denitrifiers. Most studies attempting to characterize the nosZ gene diversity using DNA-based PCR approaches have only focused on Clade I nosZ, despite recent metagenomic sequencing studies that have demonstrated the dominance of Clade II nosZ genes in many ecosystems, particularly soil. As a result, these studies have greatly underestimated the genetic potential for N2O reduction present in ecosystems. Here, we provide primer designs for seven of ten designated subclades of Clade II nosZ genes that yield suitable products for amplicon sequencing and qPCR. The diversity of Clade II NosZ made it impossible to design a universal primer set that would effectively amplify all nosZ genes in this clade. We successfully demonstrate the utility of these new primer sets in conventional PCR and amplicon sequencing. In addition, we show the utility of these primers for detecting nosZ gene transcripts from RNA extracted from soil.